Ribonuclease P RNase

Ribonuclease P (RNase P) consists of both protein and an RNA component that has catalytic activity. RNase P functions in eukaryotic cells to process the 5 end of precursor tRNA molecules. RNase P also can be directed to cleave any RNA molecule when the target is complexed with a short complementary oligonucleotide called an external guide. 

A 5 sequence of variable length that Is absent from mature tRNAs is present in all pre-tRNAs (Figure 12-36). These extra 5 nucleotides are removed by ribonuclease P (RNase P), a ribonucleoprotein endonuclease. Studies with E. coll RNase P indicate that at high Mg concentrations, the RNA component alone can recognize and cleave E. coll pre-tRNAs. The RNase P polypeptide increases the rate of cleavage by the RNA, allowing it to proceed at physiological Mg concentrations. A comparable RNase P functions In eukaryotes. 

Some nucleic acids are capable of self-splicing. These catalytic DNA and RNA are known as deoxyribozymes (Li and Breaker, 1999 Sheppard et al, 2000) and ribozymes (Doherty and Doudna, 2000 Scott and Klug, 1996) respectively. The 3 - and/or 2 -hydroxyls of DNA/RNA serve as a catalytic site that invariably requires a metal ion for the catalytic activity. Deoxyribozymes are quasi-catalytic while ribozymes can be catalytic, e.g. ribonuclease P (RNase P) as well as quasi-catalytic, e.g. introns and hammerhead RNAs. RNase P resources are maintained at http //www.mbio.ncsu.edu/RnaseP/homeJitml... 

Ribonudease P RNA. Ribonuclease P (RNase P) is the endoribonuclease that generates the mature 5 -ends of tRNA by removal of the 5 -leader elements of pre-tRNA. Haloenzymes are composed of essential RNA and protein subunits. E. coli RNase P contains a protein subunit of 119 amino acids and a single RNA molecule of 377 nucleotides, with RNA exhibiting multiple turnovers in vitro and in vivo, to process tRNA precursors with extended 5 terminal sequences (Frank and Pace, 1998 Xiao et al, 2002). Although the RNA subunit of cellular RNase P contains the catalytic active site (catalyzing maturation of tRNA in vitro), the protein subunit is required for in vivo activity. 

Ribonuclease II [EC 3.1.13.1], also called exoribo-nuclease II, catalyzes the exonucleolytic cleavage of the polynucleic acid, preferring single-stranded RNA, in the 3 - to 5 -direction to yield 5 -phosphomononucleotides. The enzyme processes 3 -terminal extra-nucleotides of monomeric tRNA precursors, following the action of ribonuclease P. Similar enzymes include RNase Q, RNase BN, RNase PHI, and RNase Y. Ribonuclease T2 [EC 3.1.27.1] is also known as ribonuclease II. 

A very different ribonuclease participates in the biosynthesis of all of the transfer RNAs of E. coli. Ribonuclease P cuts a 5 leader sequence from precursor RNAs to form the final 5 termini of the tRNAs. Sidney Altman and coworkers in 1980 showed that the enzyme consists of a 13.7-kDa protein together with a specific 377-nucleotide RNA component (designated Ml RNA) that is about five times more massive than the protein.779 Amazingly, the Ml RNA alone is able to catalyze the ribonuclease reaction with the proper substrate specificity.780 7823 The protein apparently accelerates the reaction only about twofold for some substrates but much more for certain natural substrates. The catalytic center is in the RNA, which functions well only in a high salt concentration. A major role of the small protein subunit may be to provide counterions to screen the negative charges on the RNA and permit rapid binding of substrate and release of products.783 Eukaryotes, as well as other prokaryotes, have enzymes similar to the E. coli RNase R However, the eukaryotic enzymes require the protein part as well as the RNA for activity.784... 

Ribonucleases are a widely distributed family of en-zymes that hydrolyze RNA by cutting the P—O ester bond attached to a ribose 5 carbon (fig. 8.12). A good representative of the family is the pancreatic enzyme ribonuclease A (RNase A), which is specific for a pyrimidine base (uracil or cytosine) on the 3 side of the phosphate bond that is cleaved. When the amino acid sequence of bovine RNase A was determined in 1960 by Stanford Moore and William Stein, it was the first enzyme and only the second protein to be sequenced. RNase A thus played an important role in the development of ideas about enzymatic catalysis. It was one of the first enzymes to have its three-dimensional structure elucidated by x-ray diffraction and was also the first to be synthesized completely from its amino acids. The synthetic protein proved to be enzymatically indistinguishable from the native enzyme. 

MATERIALS. Ribonuclease A (RNase A) (bovine pancreas), cytochrome C (Cyt. C) (horse heart), lysozyme (chicken egg white), myoglobin (sperm whale), P-lactoglobulin A... 

Bovine pancreatic ribonuclease A (RNase A RNA depolymerase EC 3.1.27.5) is a distributive endoribonuclease that catalyzes the cleavage of the P-O5 bond of RNA on the 3 side of pyrimidine residues. RNase A binds to polymeric substrates (Imura et al., 1965 Irie et al., 1984 Moussaoui et al., 1995), but the mechanism by which RNase A locates a pyrimidine residue within a polymeric substrate is not known. 

The 5 end is created by ribonuclease P, which cleaves to leave a phosphate on the 5 terminal G. This enzyme creates the 5 terminus of all tRNA molecules. It is not clear what structural features are recognized by RNase P, for different sequences are contained in the cleavage sites. Ribonuclease P consists of one RNA molecule of 377 nucleotides and one protein molecule with Mr of about 20,000. Both components are necessary for full catalytic activity, but under nonphysiological conditions the RNA molecule alone can... 

Ribonuclease P [Similar enzyme from RNase NU from KB cells] (3.1.26.5) is produced. [Specificity for tRNA precursors.]... 

Ribonuclease H (RNase H), also termed hybridase in some earlier literature, is an endoribonuclease that specifically degrades the RNA strand of DNA RNA heteroduplexes (Scheme 3.3). The oligoribonucleotide products have 5 -P and 3 -OH termini. 

Ribonuclease T1 (RNase Tl) is a guanine-specific endoribonuclease (also called guanyloribonuclease), which has been isolated from Aspergillus oryzae. It catalyzes the hydrolysis of phosphodiester bonds in ssRNA, producing 3 -P mononucleotides and oligonucleotides that end in Gp (Scheme 3.4). 

Many secretory proteins—e. g., pancreatic ribonuclease (RNAse see p. 74)—contain several disulfide bonds that are only formed oxidatively from SH groups after translation. The eight cysteine residues of the RNAse can in principle form 105 different pairings, but only the combination of the four disulfide bonds shown on p. 75 provides active enzyme. Incorrect pairings can block further folding or lead to unstable or insoluble conformations. The enzyme protein disulfide iso-merase [1] accelerates the equilibration between paired and unpaired cysteine residues, so that incorrect pairs can be quickly split before the protein finds its final conformation. 

Ribonucleases (RNases) may be defined as phosphodiesterases that attack the internucleotide bonds in ribonucleic acid (RNA) and its products but not those in deoxyribonucleic acid (DNA) or simple phos-phodiesters such as bis-p-nitrophenyl phosphate. 

Figure 16.12 The temperature-dependent behavior of the denaturation enthalpy and entropy of ribonuclease (RNase) and myoglobin (Mb) under the assumption that AjjjCp is constant (dashed line) or decreasing with increasing temperature (solid line). Reproduced with permission from P. L. Privalov, Ann. Rev. Biophys. Chem. 18, 47 (1989). 1989, by Annual Reviews http //www.AnnualReviews.org...

RNAs also act as ribonucleases, cleaving other RNA molecules (e.g., RNase P cleaves tRNA precursors). 

Guanyloribonuclease. Aspergillus oryzae ribonuclease. RNase Nl. Two-stage endonucleolytic cleavage to 3 -phosphomononucleotides RNase N2. and3 -phosphooligonucleotides ending in G-P with 2, 3 -cyclic... 

Soucek J, Pouckova P, Strohalm J, Plocova D, Hlouskova D, Zadinova M, Ulbrich K Poly[lY-(2-hydro rpropyl)methacry-lamide] conjugates of bovine pancreatic ribonuclease (RNase A) inhibit growth of human melanoma in nude mice. J Drug Target 2002 10 175-183. 

---